I now have several protocols to try...I'll keep you posted on how it goes.
Dina Leviten
ICOS, Corp.
Bothell, WA
> -----Original Message-----
> From: Al Cairo [SMTP:cairo@dejong.med.buffalo.edu]
> Sent: Friday, June 11, 1999 2:32 PM
> To: Leviten, Dina; ABRF
> Subject: Re: BAC sequencing
>
> Hi Dina,
> Regarding protocols, you can find our Template Preparation and Cycle
> Sequence reaction conditions for direct BAC end sequencing on the BACPAC
> Resources website. We are using 1/10 the DNA you use (200ng) and a 0.75x
> BigDye terminator reaction size(15ul) with no extra enzyme required. You
> should see consistent results and a high success rate. This is based on
> end
> sequencing many different BAC libraries produced here in Pieter deJong's
> laboratory at RPCI.
>
> You can expect 500-800 bases without any N's if you follow our methods and
> run a 48cm wtr gel on the 377 using any comb up to 64 lanes (the extent of
> our experience). From your description of the variable results problem,
> you
> may need to focus more on primer selection.
>
> Hope this helps,
> Al
> ******************
> Alfred M. Cairo
> BACPAC Resources
> DNA Sequencing Lab
> Dept. of Cancer Genetics
> Roswell Park Cancer Institute
> Buffalo NY 14263
> email: cairo@dejong.med.buffalo.edu
> web site: http://bacpac.med.buffalo.edu
> phone: 716-845-4543
>
>
> -----Original Message-----
> From: Leviten, Dina <dleviten@icos.com>
> To: Recipients of ABRF List <abrf@aecom.yu.edu>
> Date: Thursday, June 10, 1999 8:13 PM
> Subject: BAC sequencing
>
>
> >Hello All!
> >
> >What protocols are out there that describe: the preparation of the BAC
> DNA,
> >clean-up of DNA (if necessary), and the PCR reaction set-up? We could
> use
> >any advice!
> >
> >We have been sequencing directly from the BAC clone and have been getting
> >variable results. Either it works or it doesn't. Out of about different
> 50
> >primers, 12 have given good sequence. I just ran 10 primers on the 3700,
> >and 1 gave excellent sequence (700bp), 1 gave poor sequence (50bp) and
> the
> >others did not work. (This is about the same success rate as the 373S.)
> >Just so you know, we have been using 1-2ug of DNA, ~12pmols of primer and
> >adding extra enzyme. We have tried two different PCR protocols and the
> one
> >at least gives us these hit-and-miss results. We are thinking resorting
> to
> >shotgun cloning it and just sequencing the whole thing. Let me know what
> >you think...
> >
> >Thanks in advance!
> >Dina Leviten
> >ICOS, Corp.
> >Bothell, WA
> >
> >